Cd34 stem cell-related methods and compositions

ABSTRACT

This invention provides novel stem cell-based methods for treating a number of conditions. These methods employ CD34 stem cells, and have a tremendous advantage in that they do not require myeloablation in the subject being treated. The CD34 stem cells used in the instant methods can be genetically, modified or not, depending on the disorder treated.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/179,374filed Jul. 8, 2011, which is a continuation of U.S. application Ser. No.12/154,059 filed May 20, 2008, which claims priority from U.S.Provisional Patent Application Ser. No. 60/931,622 filed May 24, 2007and U.S. Provisional Patent Application Ser. No. 61/003,050 filed Nov.14, 2007.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Throughout this application, various publications are cited. Thedisclosure of these publications, as well as of the above-identifiedprovisional applications, is hereby incorporated by reference into thisapplication to describe more fully the state of the art to which thisinvention pertains.

Stem cells mediate the reproduction and transmission of geneticinformation to subsequent cellular generations. They can self-renew andgenerate differentiated progeny. In recent years progress has been madein our understanding of the molecular mechanisms that underlie theinteractions between stem cells and their tissue niches. This has led toa better understanding of the molecular regulatory mechanisms at work instem cells.

While gene therapy is still an experimental approach, the technologyholds promise for making an impact on human health. The scope anddefinition of gene therapy has changed and expanded over the past fewyears. In addition to correcting inherited genetic disorders such ascystic fibrosis, hemophilia and others entities, gene therapy approachesare also being developed to combat acquired diseases such as cancer,AIDS, chronic vascular ischemia, osteoarthritis, diabetes, Parkinson'sand Alzheimer's disease.

At present, germ line gene therapy is not being contemplated due to itscomplex technical nature and ethical considerations. However, somaticcell gene therapy exclusively for the benefit of one individual (thatcannot be passed on to succeeding generations) is a major focus of stemcell research. It has taken over 15 years of effort from the initialdescription of successful gene transfer into murine hematopoietic stemcells, to the first unambiguously successful clinical trials in patientsborn with x-linked combined immunodeficiency (SCID) and adenosinedeaminase deficiency (ADA)-deficiency(Aiuti et al., 2002;Cavazzana-Calvo et al., 2000; Gaspar et al., 2004). Many aspects of stemcell therapy are being explored. For example, retroviral vectors havebeen used in many settings for the transfer of genes into stem cells torepair mutated or incomplete genes. These include severe combined immunedeficiencies, Fanconi anemia and other hemoglobinopathies (Herzog etal., 2006).

A central issue in stem cell engineering is the specific methodologyused to introduce therapeutic genes into the progenitor cells. Becauseretroviruses tend to insert into active genes (it is thought thatcondensed chromatin opens up in these regions), it has been suggestedthat their use may also increase the risk of cancer(Young et al., 2006),because the insertion of retroviral vectors proximal to genes involvedin cell proliferation could in theory generate a precursor cancer stemcell. However, the overall risk of this type of event is difficult toestablish. There are now many examples of complete success achieved inpatients with chronic granulomatous disease (CGD) where NADPH oxidaseactivity was restored following the infusion of genetically alteredblood stem cells (Barese et al., 2004).

The minimal requirement for productive gene therapy is the sustainedproduction of the therapeutic gene product in the correct biologicalcontext with minimal harmful side effects. To achieve this end, theapplication of stem cells in genetic therapy will require thedevelopment of new strategies for modulating therapeutic geneexpression, as well as methods for the efficient delivery of foreigngenes into stem cells. The selective control of therapeutic geneexpression by differentiating stem cells within a defined tissueenvironment is an important goal in stem cell engineering. This approachcould, for example, help in the control of stem cell differentiationinto specific lineages, the maintenance of their undifferentiated statefor later transplantation, proliferation, and the regulation ofexpression of therapeutic genes such as suicide genes, cytokines orgrowth factors in defined tissue environments.

SUMMARY OF THE INVENTION

This invention provides a method for treating a subject afflicted with agastrointestinal disorder comprising introducing into the subject'sbloodstream a therapeutically effective number of CD34 stem cells,wherein (a) the CD34 stem cells are not genetically modified, (b) theintroduction of the CD34 stem cells is not preceded, accompanied orfollowed by myeloablation, and (c) the gastrointestinal disorder ischaracterized by a need for cell proliferation in the gastrointestinalendothelium.

This invention also provides a method for treating a diabetic subject ora pre-diabetic subject comprising introducing into the subject'sbloodstream a therapeutically effective number of CD34 stem cells,wherein (a) the CD34 stem cells are not genetically modified, and (b)the introduction of the CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

This invention further provides a method for treating a subjectafflicted with muscular dystrophy comprising introducing into thesubject's bloodstream a therapeutically effective number ofnon-autologous CD34 stem cells, wherein (a) the CD34 stem cells are notgenetically modified, and (b) the introduction of the CD34 stem cells isnot preceded, accompanied or followed by myeloablation.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone surgery comprising introducing into thesubject's bloodstream a therapeutically effective number of CD34 stemcells, wherein (a) the CD34 stem cells are introduced into the subject'sbloodstream immediately prior to, during, and/or immediately followingsurgery, (b) the CD34 stem cells are not genetically modified, and (c)the introduction of the CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone a physical trauma comprising introducinginto the subject's bloodstream a therapeutically effective number ofCD34 stem cells, wherein (a) the CD34 stem cells are introduced into thesubject's bloodstream immediately prior to, during, and/or immediatelyfollowing the physical trauma, (b) the CD34 stem cells are notgenetically modified, and (c) the introduction of the CD34 stem cells isnot preceded, accompanied or followed by myeloablation.

This invention provides a method for treating a subject afflicted with atumor comprising introducing into the subject's bloodstream atherapeutically effective number of genetically modified CD34 stemcells, wherein (a) each of the genetically modified CD34 stem cellscontains an exogenous nucleic acid comprising (i) a cytotoxicprotein-encoding region operably linked to (ii) a promoter orpromoter/enhancer combination, whereby the cytotoxic protein isselectively expressed when the genetically modified CD34 stem cells comeinto proximity with, and differentiate in proximity with, tumor tissueundergoing angiogenesis, and (b) the introduction of the geneticallymodified CD34 stem cells is not preceded, accompanied or followed bymyeloablation.

This invention further provides a method for treating a subjectafflicted with a gastrointestinal disorder comprising introducing intothe subject's bloodstream a therapeutically effective number ofgenetically modified CD34 stem cells, wherein (a) each of thegenetically modified CD34 stem cells contains an exogenous nucleic acidcomprising (i) a region encoding a protein which enhances endothelialcell growth, which region is operably linked to (ii) anendothelium-specific promoter or promoter/enhancer combination, (b) theintroduction of the genetically modified CD34 stem cells is notpreceded, accompanied or followed by myeloablation, and (c) thegastrointestinal disorder is characterized by a need for cellproliferation in the gastrointestinal endothelium.

This invention further provides a method for treating a diabetic subjector a pre-diabetic subject comprising introducing into the subject'sbloodstream a therapeutically effective number of genetically modifiedCD34 stem cells, wherein (a) each of the genetically modified CD34 stemcells contains an exogenous nucleic acid comprising (i) a regionencoding a protein which enhances endothelial cell growth, which regionis operably linked to (ii) an endothelium-specific promoter orpromoter/enhancer combination, and (b) the introduction of thegenetically modified CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

This invention further provides a method for treating a subjectafflicted with muscular dystrophy comprising introducing into thesubject's bloodstream a therapeutically effective number of geneticallymodified CD34 stem cells, wherein (a) each of the genetically modifiedCD34 stem cells contains an exogenous nucleic acid comprising (i) aregion encoding a protein which is absent from or under-expressed in thesubject's muscle cells or whose overexpression in the subject's musclecells is desired, which region is operably linked to (ii) amuscle-specific promoter or muscle-specific promoter/enhancercombination, and (b) the introduction of the genetically modified CD34stem cells is not preceded, accompanied or followed by myeloablation.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone surgery comprising introducing into thesubject's bloodstream a therapeutically effective number of geneticallymodified CD34 stem cells, wherein (a) each of the genetically modifiedCD34 stem cells contains an exogenous nucleic acid comprising (i) aregion encoding a protein which enhances endothelial cell growth, whichregion is operably linked to (ii) an endothelium-specific promoter orpromoter/enhancer combination, and (b) the introduction of thegenetically modified CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

Finally, this invention provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone a physical trauma comprising introducinginto the subject's bloodstream a therapeutically effective number ofgenetically modified CD34 stem cells, wherein (a) each of thegenetically modified CD34 stem cells contains an exogenous nucleic acidcomprising (i) a region encoding a protein which enhances endothelialcell growth, which region is operably linked to (ii) anendothelium-specific promoter or promoter/enhancer combination, and (b)the introduction of the genetically modified CD34 stem cells is notpreceded, accompanied or followed by myeloablation.

Other objects and features of the present invention will become apparentfrom the following detailed description considered in conjunction withthe accompanying drawings. It is to be understood, however, that thedrawings are designed solely for purposes of illustration and not as adefinition of the limits of the invention, for which reference should bemade to the appended claims. It should be further understood that thedrawings are not necessarily drawn to scale and that, unless otherwiseindicated, they are merely intended to conceptually illustrate thestructures and procedures described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1

Top row: expression of insulin in a normal murine β-islet before (left)and after alloxan (ALX) treatment (right) with almost complete insulindepletion. Bottom row: size of a normal insulin producing β-islet atdifferent magnification (20×; 40×); the treatment of insulin-depletedβ-islet after ALX treatment with CD34-negative stem cells (SC)completely restores insulin production with signs of hypertrophy (20×and 40×).

FIG. 2

Isolated cells from a murine pancreas after ALX-induced diabetes and SCrestoration of insulin production. The transplanted CD34-negative stemcells were marked with a constitutively expressed green fluorescenceprotein, and the insulin-producing cells showed a red fluorescence.There was no co-expression of both markers, suggesting that thetransplanted stem cells do not express insulin by themselves but ratherfacilitate the endogenous regeneration.

FIG. 3

Left: Blood glucose levels of mice after ALX treatment without SCtransplantation (top), with SC transplantation and no correction of theblood glucose level (middle) and mice with a normalized blood glucoselevel after SC transplantation (bottom). Right: Only mice that revealedthe presence of stem cells in their pancreas (homogenized for FACSanalysis and detection of green fluorescence) (E3; red circle) showed anormalized blood glucose level, suggesting the pivotal role oftransplanted cells in the correction of insulin production.

FIG. 4

Schematic presentation of the development of an epithelial malignancyfrom an in-situ carcinoma to invasive cancer and the connection to theendogenous blood vessel system. CD34-negative stem cells home to a siteof neo-angiogenesis as demonstrated here and can therefore be utilizedas a Trojan Horse to deliver cytotoxic or immune modulatory agents.

FIG. 5

Detection RFP-positive cells in mammary tumors. Tie2-RFP transfectedstem cells differentiate to endothelial and transcribe the RFP. (A)Counterstained with DAPI. (B) RFP-positive cells forming vessels.

FIG. 6

Reduced tumor progression under GCV treatment. (A) Stem cell-GCVapplication protocol. The cell suspension (day 0) and the GCV-solution(day 5-8) respectively were applied as shown. Increase of bodyweightduring the treatment of mice reflected the total tumor load as allbreasts were involved. Body weight was measured on day 0 and 5 of eachcycle of therapy and the day of dissection. (B) Groups of mice,treatment starting in week 22, average showing standard deviation. Micewere sorted in one treatment group and two control groups. First controlgroup received 1xPBS instead of a stem cell suspension and nodrug-injection (dashed line); the second control group received a stemcell suspension transfected with Tie2-RFP but no GCV (dotted line). Thetreatment group received stem cells and GCV as shown in A, (solid line).(C) Groups of treatment starting in week 18, average and standarddeviation.

FIG. 7

Age at dissection. Controls vs. treatment group of mice startingtreatment at week 22. Note the significant difference in time to reachsimilar tumor sizes (see table 1) and the extended life span of miceafter successful treatment with MSC TK-vehicles and GCV.

FIG. 8

Tumor regrowth model: Primary breast tumor was resected at 18 weeks andMSC/tk treatment was initiated during regrowth of the tumor.

FIG. 9

MSC—expressing green florescent protein (GFP) home to the growingpancreatic tumor. In parallel experiments, MSC engineered to express redflorescent protein (RFP) under the control of the Tie2 promoter/enhancershow a directed expression in the tumor vasculature. Top row: MSCengineered to express GFP under the control of the CMV promoter home tothe tumor following i.v. injection. Bottom row: MSC engineered toexpress RFP under the control of the Tie2.

FIG. 10

The effect of the tk/GCV treatment was then assessed after the injectionof the C57B1/6 MSC (Tie2-tk) cells.

The treatment regimen was essentially as described for the breast cancerstudy. 500,000 cells were injected on day one, followed by three dayswhere the cells were allowed to be recruited to the growing tumor and todifferentiate into endothelial-like Tie2 expressing cells thusexpressing the TK suicide gene. The mice were then treated for four dayswith GVC. After one day of rest, the cycle repeated for the duration ofthe experiment.

FIG. 11

Figure shows an additional example of the effect of treatment of theorthotopic pancreatic tumor with therapeutic stem cells together withGCV. A dramatic reduction in tumor size (50%) as well as reducedperitoneal carcinosis was seen in comparison to the untreated group.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Terms

In this application, certain terms are used which shall have themeanings set forth as follows.

As used herein, “acute wound healing” shall include, without limitation,a cellular and molecular process which is activated to repair tissuefrom the moment of injury under the control of biological and mechanicalsignals. Successful acute wound healing occurs when a dynamic balance ismet between the loads placed across a provisional matrix and thefeedback of repair cells.

As used herein, a cell is “allogenic” with respect to a subject if it orany of its precursor cells are from another subject of the same species.

As used herein, a cell is “autologous” with respect to a subject if itor its precursor cells are from that same subject.

As used herein, “CD34 stem cell” shall mean a stem cell lacking CD34 onits surface. CD34 stem cells, and methods for isolating same, aredescribed, for example, in Lange C. et al., Accelerated and safeexpansion of human mesenchymal stromal cells in animal serum-free mediumfor transplantation and regenerative medicine. J. Cell Physiol. 2007,Apr. 25 [Epub ahead of print].

As used herein, “cell proliferation” shall mean the division, growth insize and/or differentiation of cells.

As used herein, “cytotoxic protein” shall mean a protein which, whenpresent in, on and/or in proximity with a cell, causes that cell's deathdirectly and/or indirectly. Cytotoxic proteins include, for example,suicide proteins (e.g. HSV-tk) and apoptosis inducers. Cytotoxic genesinclude null genes, siRNA or miRNA for gene knockdown (e.g. CCR5−/−). Anumber of suicide gene systems have been identified, including theherpes simplex virus thymidine kinase gene, the cytosine deaminase gene,the varicella-zoster virus thymidine kinase gene, the nitroreductasegene, the Escherichia coli gpt gene, and the E. coli Deo gene. Cytosinedeaminase; Cytochrome P450; Purine nucleoside phosphorylase;Carboxypeptidase G2; Nitroreductase. As detailed in: Yazawa K, Fisher WE, Brunicardi F C: Current progress in suicide gene therapy for cancer.World J Surg. 2002 July; 26(7):783-9. Cytotoxic factors include thefollowing: (i) homing factors such as chemokines and mucin chemokine GPIfusions (chemokine derived agents can be used to facilitate the directedrecruitment of engineered stem cells, see, e.g., PCT InternationalApplication No. PCT/EP2006/011508, regarding mucin fusions anchored withGPI); (ii) viral antigens (measles, chicken pox) as cytotoxic proteins;and (iii) Her2/neu antigens which can be presented on the surfaces ofengineered stem cells, followed by administration of her-2/neu antibody,and CamPath® (Alemtuzumab) directed against a CD52 epitope.

As used herein, “endothelial cell” shall include, without limitation, acell that forms the inner lining of the intima in blood vessels duringor after a process called angiogenesis. The factors controlling thisprocess are called angiogenic factors.

Endothelial cells also act with circulating blood cells by means ofreceptor-ligand interactions.

As used herein, an “endothelium-specific promoter or promoter/enhancercombination” is a promoter or promoter/enhancer combination,respectively, which when in an endothelial cell in or in proximity withendothelial cells, causes expression of an operably linked encodingregion more than it would in any other milieu in the subject.

As used herein, a nucleic acid is “exogenous” with respect to a cell ifit has been artificially introduced into that cell or any of that cell'sprecursor cells.

As used herein, “gastrointestinal disorder” shall mean any disorder ofthe stomach, small intestine and/or large intestine.

As used herein, a stem cell is “genetically modified” if either it orany of its precursor ceils have had nucleic acid artificially introducedthereinto. Methods for generating genetically modified stem cellsinclude the use of viral or non-viral gene transfer (e.g., plasmidtransfer, phage integrase, transposons, AdV, AAV and Lentivirus).

As used herein, “immediately prior to” an event includes, for example,within 5, 10 or 30 minutes prior to, or 1, 2, 6, 12 or 24 hours prior tothe event. “Immediately following” an event includes, for example,within 5, 10 or 30 minutes after, or 1, 2, 6, 12 or 24 hours after tothe event.

As used herein, “integration” of a nucleic acid into a cell can betransient or stable.

As used herein, “introducing” CD34 stem cells “into the subject'sbloodstream” shall include, without limitation, introducing such cellsinto one of the subject's veins or arteries via injection. Suchadministering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods. A single injection ispreferred, but repeated injections overtime (e.g., quarterly,half-yearly or yearly) may be necessary in some instances. Suchadministering is also preferably performed using an admixture of CD34stem cells and a pharmaceutical̂ acceptable carrier. Pharmaceuticallyacceptable carriers are well known to those skilled in the art andinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers can be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions and suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's and fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers such as Ringer'sdextrose, those based on Ringer's dextrose, and the like. Fluids usedcommonly for i.v. administration are found, for example, in Remington:The Science and Practice of Pharmacy, 20^(th) Ed., p. 808, LippincottWilliams & Wilkins (2000). Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, inert gases, and the like.

As used herein, “microcirculation” shall include, without limitation,the flow of blood from aterioles to capillaries or sinusoids to venules.Under certain circumstances, the term microcirculation is also appliedto lymphatic vessels.

As used herein, “myeloablation” shall mean the severe or completedepletion of bone marrow cells caused by, for example, theadministration of high doses of chemotherapy or radiation therapy.Myeloablation is a standard procedure and is described, for example, inDeeg H J, Klingemann H G, Philips G L, A Guide to Bone MarrowTransplantation. Springer-Verlag Berlin Heidelberg 1992.

As used herein, a stem cell is “not genetically modified” if neither itnor any of its precursor cells have had nucleic acid artificiallyintroduced thereinto.

As used herein, “nucleic acid” shall mean any nucleic acid molecule,including, without limitation, DNA, RNA and hybrids thereof. The nucleicacid bases that form nucleic acid molecules can be the bases A, C, G, Tand U, as well as derivatives thereof. Derivatives of these bases arewell known in the art, and are exemplified in PCR Systems, Reagents andConsumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems,Inc., Branchburg, N.J., USA).

As used herein, a cytotoxic protein-encoding nucleic acid region is“operably linked” to a promoter or promoter/enhancer combination if suchpromoter or promoter/enhancer combination causes the expression of thecytotoxic protein.

As used herein, a “polypeptide” means a polymer of amino acid residues.A “peptide” typically refers to a shorter polypeptide (e.g., 10 aminoacid residues), and a “protein” typically refers to a longer polypeptide(e.g., 200 amino acid residues). The amino acid residues can benaturally occurring or chemical analogues thereof. Polypeptides can alsoinclude modifications such as glycosylation, lipid attachment,sulfation, hydroxylation, and ADP-ribosylation.

As used herein, a “prediabetic” subject includes, without limitation, asubject who has the complex of symptoms that indicate he will likelydevelop insulin-dependent diabetes. Prediabetic subjects have ahigher-than-normal insulin levels.

As used herein, a “promoter” includes, without limitation, endothelin-1promoter, pre-proendothelin-1 promoter, myoD promoter, NeuroD promoter,CD20 promoter, insulin promoter, Pdx-1 promoter, VEGF promoter, VEGF-Rpromoter, SCL promoter, Seal promoter, BDNF(-R) promoter, NGF(-R)promoter and EGF-R promoter.

As used herein, “promoter/enhancers” include, without limitation, Tie2promoter enhancer, and Flk1 promoter and intronic enhancer.

As used herein, in “proximity with” a tissue includes, for example,within 1 mm of the tissue, within 0.5 mm of the tissue and within 0.25mm of the tissue.

As used herein, a cytotoxic protein is “selectively expressed” when agenetically modified CD34 stem cell encoding same comes into proximitywith, and differentiates in proximity with, tumor tissue undergoingangiogenesis, if the cytotoxic protein is expressed in that milieu morethan it is expressed in any other milieu in the subject. Preferably, thecytotoxic protein is expressed in that milieu at least 10 times morethan it is expressed in any other milieu in the subject.

As used herein, “subject” shall mean any animal, such as a human,non-human primate, mouse, rat, guinea pig or rabbit.

As used herein, a “therapeutically effective number of CD34 stem cells”includes, without limitation, the following amounts and ranges ofamounts: (i) from about 1×10² to about 1×10⁸ cells/kg body weight; (ii)from about 1×10³ to about 1×10⁷ cells/kg body weight; (iii) from about1×10⁴ to about 1×10⁶ cells/kg body weight; (iv) from about 1×10⁴ toabout 1×10⁵ cells/kg body weight; (v) from about 1×10⁵ to about 1×10⁶cells/kg body weight; (vi) from about 5×10⁴ to about 0.5×10⁵ cells/kgbody weight; (vii) about 1×10³ cells/kg body weight; (viii) about 1×10⁴cells/kg body weight; (ix) about 5×10⁴ cells/kg body weight; (x) about1×10⁵ cells/kg body weight; (xi) about 5×10⁵ cells/kg body weight; (xii)about 1×10⁶ cells/kg body weight; and (xiii) about 1×10⁷ cells/kg bodyweight. Human body weights envisioned include, without limitation, about50 kg, about 60 kg; about 70 kg; about 80 kg, about 90 kg; and about 100kg. These numbers are based on pre-clinical animal experiments andstandard protocols from the transplantation of CD34+ hematopoietic stemcells. Mononuclear cells (including CD34⁺ cells) usually contain between1:23,000 to 1:300,000 CD34 cells.

As used herein, “treating” a subject afflicted with a disorder shallmean slowing, stopping or reversing the disorder's progression. In thepreferred embodiment, treating a subject afflicted with a disorder meansreversing the disorder's progression, ideally to the point ofeliminating the disorder itself. As used herein, ameliorating a disorderand treating a disorder are equivalent.

As used herein, “tumor” shall include, without limitation, avascularized tumor such as a prostate tumor, a pancreatic tumor, asquamous cell carcinoma, a breast tumor, a melanoma, a basal cellcarcinoma, a hepatocellular carcinoma,

testicular cancer, a neuroblastoma, a glioma or a malignant astrocytictumor such as glioblastma multiforme, a colorectal tumor, an endometrialcarcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, anosteosarcoma, a rhabdo/leiomyosarcoma, a synovial sarcoma, anangiosarcoma, an Ewing sarcoma/PNET and a malignant lymphoma. Theseinclude primary tumors as well as metastatic diseases.

As used herein, a cell is “xenogenic” with respect to a subject if it orany of its precursor cells are from another subject of a differentspecies.

Embodiments of the Invention

This invention provides novel stem cell-based methods for treatingcertain conditions. These methods employ CD34 stem cells, as opposed tolater stage stem cells, and have a tremendous advantage in that they donot require myeloablation for the subject being treated. The CD34 stemcells used in the instant methods can be genetically modified or not,depending on the disorder treated. The instant methods are detailedbelow, beginning with methods employing non-genetically modified CD34stem cells and followed by methods employing genetically modified CD34stem cells.

Specifically, this invention provides a method for treating a subjectafflicted with a gastrointestinal disorder comprising introducing intothe subject's bloodstream a therapeutically effective number of CD34stem cells, wherein (a) the CD34 stem cells are not geneticallymodified, (b) the introduction of the CD34 stem cells is not preceded,accompanied or followed by myeloablation, and (c) the gastrointestinaldisorder is characterized by a need for cell proliferation in thegastrointestinal endothelium.

In this method, the gastrointestinal disorder includes, withoutlimitation, colitis, ulcerative colitis, inflammatory bowel disorder,Crohn's disease, colitis due to acute and chronic intestinal ischemia,celiac disease, Whipple disease, or Graft-versus-Host disease after stemcell transplantation.

This invention also provides a method for treating a diabetic subject ora pre-diabetic subject comprising introducing into the subject'sbloodstream a therapeutically effective number of CD34 stem cells,wherein (a) the CD34 stem cells are not genetically modified, and (b)the introduction of the CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

In one embodiment of this method, the subject is pre-diabetic, eitherfor type I diabetes or type II diabetes. In another embodiment, thesubject is diabetic, afflicted either with type I diabetes or type IIdiabetes.

This invention further provides a method for treating a subjectafflicted with muscular dystrophy comprising introducing into thesubject's bloodstream a therapeutically effective number ofnon-autologous CD34 stem cells, wherein (a) the CD34 stem cells are notgenetically modified, and (b) the introduction of the CD34 stem cells isnot preceded, accompanied or followed by myeloablation.

In the preferred embodiment of this method, the subject is afflictedwith Duchenne or Becker's muscular dystrophy, and the CD34 stem cellsare allogenic with respect to the subject.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone surgery comprising introducing into thesubject's bloodstream a therapeutically effective number of CD34 stemcells, wherein (a) the CD34 stem cells are introduced into the subject'sbloodstream immediately prior to, during, and/or immediately followingsurgery, (b) the CD34 stem cells are not genetically modified, and (c)the introduction of the CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

This method is appropriate for any type of surgery including, withoutlimitation, abdominal surgery, thoracic surgery, neurosurgery, plasticsurgery or trauma surgery. Additionally, the surgery can be laproscopicsurgery or open surgery.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone a physical trauma comprising introducinginto the subject's bloodstream a therapeutically effective number ofCD34 stem cells, wherein (a) the CD34 stem cells are introduced into thesubject's bloodstream immediately prior to, during, and/or immediatelyfollowing the physical trauma, (b) the CD34 stem cells are notgenetically modified, and (c) the introduction of the CD34 stem cells isnot preceded, accompanied or followed by myeloablation.

This method is appropriate for any type of physical trauma. Specificallyenvisioned are (i) childbirth, wherein the CD34 stem cells areintroduced into the subject's bloodstream immediately prior to, duringor immediately following the event,(ii) a flesh wound caused by aviolent act, wherein the CD34 stem cells are introduced into thesubject's bloodstream immediately following the physical trauma, and(iii) a burn wound, wherein the CD34 stem cells are introduced into thesubject's bloodstream immediately following the physical trauma.

In the above methods employing non-genetically modified CD34 stem cells,the subject treated can be any subject. In the preferred embodiment, thesubject is human. Furthermore, in the subject methods employingnon-genetically modified CD34 stem cells, the CD34 stem cells can beallogenic, autologous or xenogenic with respect to the subject, unlessstated or implied otherwise.

This invention provides a method for treating a subject afflicted with atumor comprising introducing into the subject's bloodstream atherapeutically effective number of genetically modified CD34 stemcells, wherein (a) each of the genetically modified CD34 stem cellscontains an exogenous nucleic acid comprising (i) a cytotoxicprotein-encoding region operably linked to (ii) a promoter orpromoter/enhancer combination, whereby the cytotoxic protein isselectively expressed when the genetically modified CD34 stem cells comeinto proximity with, and differentiate in proximity with, tumor tissueundergoing angiogenesis, and (b) the introduction of the geneticallymodified CD34 stem cells is not preceded, accompanied or followed bymyeloablation.

All tumor types are envisioned for this method including, for example, aprostate tumor, a pancreatic tumor, a squamous cell carcinoma, a breasttumor, a melanoma, a basal cell carcinoma, a hepatocellular carcinoma,testicular cancer, a neuroblastoma, a glioma or a malignant astrocytictumor such as glioblastma multiforme, a colorectal tumor, an endometrialcarcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, anosteosarcoma, a rhabdo/leiomyosarcoma, a synovial sarcoma, anangiosarcoma, an Ewing sarcoma/PNET and a malignant lymphoma.

Numerous promoter/enhancer combinations and cytotoxic proteins are alsoenvisioned for this method. In one embodiment, the promoter/enhancercombination is the Tie2 promoter/enhancer, the cytotoxic protein isHerpes simplex viral thymidine kinase, and the subject is treated withGanciclovir® in a manner permitting the Herpes simplex viral thymidinekinase to render the Ganciclovir® cytotoxic. Ganciclovir® and itsmethods of use are well known in the art.

This invention further provides a method for treating a subjectafflicted with a gastrointestinal disorder comprising introducing intothe subject's bloodstream a therapeutically effective number ofgenetically modified CD34 stem cells, wherein (a) each of thegenetically modified CD34 stem cells contains an exogenous nucleic acidcomprising (i) a region encoding a protein which enhances endothelialcell growth, which region is operably linked to (ii) anendothelium-specific promoter or promoter/enhancer combination, (b) theintroduction of the genetically modified CD34 stem cells is notpreceded, accompanied or followed by myeloablation, and (c) thegastrointestinal disorder is characterized by a need for cellproliferation in the gastrointestinal endothelium.

In this method, the gastrointestinal disorder is preferably colitis,ulcerative colitis, inflammatory bowel disorder or Crohn's disease.

Numerous promoter/enhancer combinations and endothelial cellgrowth-enhancing proteins are envisioned for this method. In oneembodiment, the promoter/enhancer combination is the Tie2promoter/enhancer and the protein which enhances endothelial cell growthis a vascular endothelial growth factor (VEGF). Other angiogenic factorsin addition to VEGF are also envisioned, such as HIF-1a andCarboanhydrase IX.

This invention further provides a method for treating a diabetic subjector a pre-diabetic subject comprising introducing into the subject'sbloodstream a therapeutically effective number of genetically modifiedCD34 stem cells, wherein (a) each of the genetically modified CD34 stemcells contains an exogenous nucleic acid comprising (i) a regionencoding a protein which enhances endothelial cell growth, which regionis operably linked to (ii) an endothelium-specific promoter orpromoter/enhancer combination, and (b) the introduction of thegenetically modified CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

In one embodiment of this method, the subject is pre-diabetic, eitherfor type I diabetes or type II diabetes. In another embodiment, thesubject is diabetic, afflicted either with type I diabetes or type IIdiabetes.

Numerous promoter/enhancer combinations and endothelial cellgrowth-enhancing proteins are envisioned for this method. In oneembodiment, the promoter/enhancer combination is the Tie2promoter/enhancer and the protein which enhances endothelial cell growthis a vascular endothelial growth factor (VEGF) associated withangiogenesis.

This invention further provides a method for treating a subjectafflicted with muscular dystrophy comprising introducing into thesubject's bloodstream a therapeutically effective number of geneticallymodified CD34 stem cells, wherein (a) each of the genetically modifiedCD34 stem cells contains an exogenous nucleic acid comprising (i) aregion encoding a protein which is absent from or under-expressed in thesubject's muscle cells or whose overexpression in the subject's musclecells is desired, which region is operably linked to (ii) amuscle-specific promoter or muscle-specific promoter/enhancercombination, and (b) the introduction of the genetically modified CD34stem cells is not preceded, accompanied or followed by myeloablation.

In the preferred embodiment of this method, the subject is afflictedwith Duchenne or Becker's muscular dystrophy, and the CD34 stem cellsare allogenic or autologous with respect to the subject.

Numerous muscle-specific promoter/enhancer combinations are envisionedfor this method. In one embodiment, the muscle-specificpromoter/enhancer combination is the MyoD promoter/enhancer. In thepreferred embodiment of Duchenne muscular dystrophy, the protein absentfrom the subject's muscle cells is dystrophin.

This invention further provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone surgery comprising introducing into thesubject's bloodstream a therapeutically effective number of geneticallymodified CD34 stem cells, wherein (a) each of the genetically modifiedCD34 stem cells contains an exogenous nucleic acid comprising (i) aregion encoding a protein which enhances endothelial cell growth, whichregion is operably linked to (ii) an endothelium-specific promoter orpromoter/enhancer combination, and (b) the introduction of thegenetically modified CD34 stem cells is not preceded, accompanied orfollowed by myeloablation.

This method is appropriate for any type of surgery including, withoutlimitation, abdominal surgery, thoracic surgery, neurosurgery or plasticsurgery. Additionally, the surgery can be laproscopic surgery or opensurgery.

Numerous promoter/enhancer combinations and endothelial cellgrowth-enhancing proteins are envisioned for this method. In oneembodiment, the promoter/enhancer combination is the Tie2promoter/enhancer and the protein which enhances endothelial cell growthis a vascular endothelial growth factor (VEGF) associated withangiogenesis.

Finally, this invention provides a method for improving microcirculationand/or acute wound healing in a subject who is about to undergo, isundergoing or has undergone a physical trauma comprising introducinginto the subject's bloodstream a therapeutically effective number ofgenetically modified CD34 stem cells, wherein (a) each of thegenetically modified CD34 stem cells contains an exogenous nucleic acidcomprising (i) a region encoding a protein which enhances endothelialcell growth, which region is operably linked to (ii) anendothelium-specific promoter or promoter/enhancer combination, and (b)the introduction of the genetically modified CD34 stem cells is notpreceded, accompanied or followed by myeloablation.

This method is appropriate for any type of physical trauma. Specificallyenvisioned are (i) childbirth, (ii) a flesh wound caused by a violentact, wherein the CD34 stem cells are introduced into the subject'sbloodstream immediately following the physical trauma, and (iii) a burnwound, wherein the CD34 stem cells are introduced into the subject'sbloodstream immediately following the physical trauma.

Numerous promoter/enhancer combinations and endothelial cellgrowth-enhancing proteins are envisioned for this method. In oneembodiment, the promoter/enhancer combination is the Tie2promoter/enhancer and the protein which enhances endothelial cell growthis a vascular endothelial growth factor (VEGF) associated withangiogenesis.

In the above methods employing genetically modified CD34 stem cells, thesubject treated can be any subject. In the preferred embodiment, thesubject is human. Furthermore, in the subject methods employinggenetically modified CD34 stem cells, the CD34 stem cells can beallogenic, autologous or xenogenic with respect to the subject, unlessstated or implied otherwise.

In the instant methods employing genetically modified CD34 stem cells,the exogenous genes are expressed, i.e., “turned on”, when the stemcells (i) come into proximity with the appropriate cells in targettissue, (ii) differentiate, and/or (iii) fuse with the appropriate cellsin target tissue.

The various proteins and regulatory sequences used in this invention canbe readily obtained by one skilled in the art. For example, endothelialcell specificity of the Tie2 promoter enhancer is shown in Schlaeger TM, Bartunkova S, Lawitts J A, Teichmann G, Risau W, Deutsch U, Sato T N.Uniform vascular-endothelial-cell-specific gene expression in bothembryonic and adult transgenic mice. Proc Natl Acad Sci USA. 199794:3058-63. The HSV TK—V00467 Herpes gene can be used for thymidinekinase (ATP:thymidine 5′ phosphotransferase, e.c. 2.7.1.21) (type 1strain CL101).

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details Part I Genetically Engineered TransgenicCD34-Negative Stem Cells for Therapeutic Gene Delivery Synopsis

Stem cell and gene therapy approaches hold much hope for the developmentof new tools to treat many life-threatening diseases. The linking ofstem cell therapy with selective gene therapy enhances therapeuticoptions for the regeneration or replacement of diseased or missingcells. Tissue-specific gene expression in the context of differentiationof CD34-negative, in-vitro adherent growing stem cells are used togenerate transgenic CD34-negative progenitor cells, which will lead toselectivity of cells and inducibility of gene expression also for safetyreasons. Viral and non-viral genes delivering technologies are detailedas are techniques for the modulation of gene expression in the contextof stem cell recruitment and differentiation. Potential clinicalapplications for this new therapeutic strategy are described, bringingthe transgenic progenitor cells to the cancer or site of tissueregeneration to induce antitumor therapy or promote tissue remodelingand wound healing. Transgenic progenitor cells serve as potent genedelivery vehicle.

Stem Cells as Gene Delivery Vehicles

Stem cells offer the potential to provide cellular therapies fordiseases that are refractory to other treatments. For each type of stemcell the ultimate goal is the same: the cell should express a specificrepertoire of genes, thereby modifying cell identity to maintain,replace, or rescue a particular tissue. To help support differentiationin the specific tissue environment attempts are being made to modify the“nuclear programming” of stem cells.

Multipotent stem cells, mesenchymal stem cells and multipotent adultprogenitor cells (MAPCs) represent promising stem cell populations asthey are capable of differentiating along different lineages. Theyrepresent the “cellular engines” that drive the renewal of adultmammalian tissues. These cells divide continuously throughout life toproduce new progeny cells that undergo a program of differentiation andmaturation to replace older expired tissue cells. The same cell turnoverprogram is thought in some cases to provide a source of cells for therepair and regeneration of adult tissues. The regenerative potential ofthe different stem cell types underlies the current interest in adaptingthese cells for applications in cell replacement therapy.

Potential sources of stem cells for therapy include bone marrow,peripheral blood, CNS, liver, pancreas, muscle, skin lung, intestineheart and fat (Koerbling M, Estrov Z, Adult stem cells for tissuerepair-anew therapeutic concept?) NEJM 2003 349: 570-582). For clinicalapplication the sources of stem cells should be easily accessible andreadily harvested with minimal risk to the patient and provide abundantcells. In this regard fat tissue represents a promising tissue source.(Adipose derived stem cells for the regeneration of damaged tissueParker M, Adam K, Expert Opion Biol Therap, 2006, 567-568) Adiposederived stem cells and bone marrow derived stem cells share similargrowth kinetics, characteristics regarding cell senescence, genetransduction efficiency, CD surface marker expression and genetranscription profiles (Cells Tissues Organs. 2003; 174(3): 101-9.Comparison of multi-lineage cells from human adipose tissue and bonemarrow. De Ugarte D A, Morizono K, Elbarbary A, Alfonso Z, Zuk P A, ZhuM, Dragoo J L, Ashjian P, Thomas B, Benhaim P, Chen I, Fraser J, HedrickM H, Mol Biol Cell. 2002 December; 13(12):4279-95. Human adipose tissueis a source of multipotent stem cells. Zuk P A, Zhu M, Ashjian P, DeUgarte D A, Huang J I, Mizuno H, Alfonso Z C, Fraser J K, Benhaim P,Hedrick M H).

Stem cells derived from different sources are also being evaluated aspotential vehicles for cell and gene specific therapy against disease.Their high self-renewal potential makes them promising candidates forthe restoration or replacement of organ systems and/or the delivery ofgene products. While progenitor cells may show good proliferation anddifferentiation potential in vitro, their biological properties in vivoremain to be defined. Stem cells expanded in vitro representheterogeneous populations that include multiple generations ofmesenchymal (stromal) cell progeny, which lack the expression of mostdifferentiation markers like CD34. These populations may have retained alimited proliferation potential and responsiveness for terminaldifferentiation and maturation along mesenchymal and non-mesenchymallineages. Hopefully in the future better markers for multipotent stemcell populations will improve the ability to distinguish these stemcells from other progenitor cell populations.

Tissue-Specific Promoters Used to Deliver Therapeutic Gene Expression inthe Correct Biological Context.

Stem-cell-mediated therapy ultimately entails nuclear reprogramming—thealteration of gene expression patterns unique to cell types in diversetissues and organs. In a number of inherited stem cell diseases, agenetic defect imparts a survival disadvantage to the affected stem cellpopulation. In these diseases, the transplantation of a “corrected” stemcell population is thought to undergo spontaneous in vivo selection inthe absence of any exogenously applied selective pressure. For example,in X-linked SCID the introduction of a therapeutic transgene confers acontinuous proliferation and survival advantage to the transduced cellpopulation (Neff et al., 2006). However, similar in vivo selectioneffects are usually not directly possible in a majority of diseases. Insettings where an over expression of the therapeutic gene does notconfer a survival advantage, a second selectable gene, ideally underpharmacological regulation, can be incorporated into the vector. (Tiranaand Kim, 2005). Systems that allow for pharmacologically regulatedselection include reversible forced protein-protein interaction using socalled “chemical inducers of dimerization” (CIDs). These systems rely ontwo components. The first is a ligand or drug, and the second is afusion protein that combines a ligand-binding protein domain and aneffector domain (usually the intracellular proportion of a growth factorreceptor). The effector domain is activated by drug binding leading toprotein dimerization. The signaling fusion thus serves as a switch thatis turned on in the presence of the CID and off following withdrawal ofthe CID. The incorporation of systems like this into a stem cellpopulation can allow a drug-dependent control of proliferation of thetransduced cell population(Neff et al., 2006; Neff and Blau, 2001).

The use of stem cells as delivery vehicles for therapeutic genes can beseen to offer a series of advantages. Stem cells are often activelyrecruited to damaged tissues where they undergo differentiation duringtissue repair. For example CD34+ bone marrow-derived progenitor cellscontribute to tissue repair by differentiating into endothelial cells,vascular smooth muscle cells, hematopoietic cells, and possibly othercell types. However, the mechanisms by which circulating progenitorcells home to remodeling tissues remain unclear. Jin et al. havedemonstrated that integrin α4β1 (VLA-4) can promote the homing ofcirculating progenitor cells to the α4β1 ligands VCAM and cellularfibronectin expressed on actively remodeling neovasculature. Progenitorcells which express integrin α4β1 were shown to home to sites of activetumor neovascularization but not to normal tissues. Antagonists ofintegrin α4β1, but not other integrins, blocked the adhesion of thesecells to endothelia and outgrowth into differentiated cell types. (Jinet al, 2006)

In addition to integrins, chemokines and their receptors also appear toplay central roles in the tissue-specific homing of stem cells. On thebasis of their chemokine receptor expression profile, CD34 MSCs werepredicted to home to secondary lymphatic organs (CCR7), skin (CCR4,CCR10), small intestine (CCR10), and salivary glands (CCR10). Aftertransiently labeling CD34− MSC with CMFDA or stably introducing greenfluorescent protein (GFP) expression plasmids, the cells were injectedinto syngeneic healthy mice and the tissue distribution of the cellsdetermined one three and seven days later. Interestingly, the stem cellsdid not home back to the bone marrow but were found to migrate tosecondary lymphatic organs, salivary glands, intestine and skin inaccordance with their chemokine receptor expression profile.

Given that stem cells can show a selective migration to different tissuemicroenvironments in normal as well as diseased settings, the use oftissue-specific promoters linked to the differentiation pathwayinitiated in the recruited stem cell could in theory be used to drivethe selective expression of therapeutic genes only within a definedbiologic context. Stem cells that are recruited to other tissue niches,but do not undergo the same program of differentiation, should notexpress the therapeutic gene. This approach allows a significant degreeof potential control for the selective expression of the therapeuticgene within a defined microenvironment and has been successfully appliedto regulate therapeutic gene expression during neovascularization.

A large number of promoters have been characterized for theirtissue-specific expression. A good source for this information can befound in the transgene literature, or for example in the variousdatabases that list tissue-specific promoter activity for the expressionof the CRE transgenes used to drive tissue-specific CRE/Lox targetedgene deletion models in mouse (for example:http://www.mshri.on.ca/nagy/Cre-works.htm). Promoters can be introducedthat are selectively regulated in the context of inflammation orneovascularization. In this regard the Tie2-promoter, Flkl promoter andintronic enhancer, endothelin-1 promoter and the pre-proendothelin-1promoter have been studied for endothelial specific expression(Huss etal., 2004). The application of specific reporter genes and new imagingtechniques can be used to define the tissue-specific expression of thecandidate promoter within the context of stem cell transplantation.Other options regarding the delivery of genes include the application ofan internal ribosome entry site (IRES) signal for the expression ofmultiple genes from a single promoter(Jackson, 2005), for example, atherapeutic gene in conjunction with a reporter gene can be used tobetter follow the distribution of expression of the therapeutic gene inan experimental context.

Importantly, many promoters can show “leakage” of expression in othertissue types or a low level basal expression in the engineered cells.Promoter engineering is a new technology that can allow one to “tune”promoter specificity to limit cross tissue activity thus allowing a morerestrictive expression to specific cell types(Fessele et al., 2002;Werner et al., 2003).

Gene Delivery Methods

The various gene delivery methods currently being applied to stem cellengineering include viral and non viral vectors, as well as biologicalor chemical methods of transfection. The methods can yield either stableor transient gene expression in the system used.

Viral Gene Delivery Systems

Because of their high efficiency of transfection, genetically modifiedviruses have been widely applied for the delivery of genes into stemcells.

DNA Virus Vectors

-   -   (i) Adenovirus

Adenoviruses are double stranded, nonenveloped and icosahedral virusescontaining a 36 kb viral genome(Kojaoghlanian et al., 2003). Their genesare divided into early (E1A, E1B, E2, E3, E4), delayed (IX, IVa2) andmajor late (L1, L2, L3, L4, L5) genes depending on whether theirexpression occurs before or after DNA replication. To date, 51 humanadenovirus serotypes have been described which can infect and replicatein a wide range of organs. The viruses are classified into the followingsubgroups: A—induces tumor with high frequency and short latency, B—areweakly oncogenic, and C—are non-oncogenic (Cao et al., 2004;Kojaoghlanian et al., 2003). These viruses have been used to generate aseries of vectors for gene transfer cellular engineering. The initialgeneration of adenovirus vectors were produced by deleting the E1 gene(required for viral replication) generating a vector with a 4 kb cloningcapacity. An additional deletion of E3 (responsible for host immuneresponse) allowed an 8 kb cloning capacity(Bett et al., 1994; Danthinneand Imperiale, 2000; Danthinne and Werth, 2000). The second generationof vectors was produced by deleting the E2 region (required for viralreplication) and/or the E4 region (participating in inhibition of hostcell apoptosis) in conjunction with E1 or E3 deletions. The resultantvectors have a cloning capacity of 10-13 kb(Armentano et al., 1995). Thethird “gutted” generation of vectors was produced by deletion of theentire viral sequence with the exception of the inverted terminalrepeats (ITRs) and the cis acting packaging signals. These vectors havea cloning capacity of 25 kb (Kochanek et al., 2001) and have retainedtheir high transfection efficiency both in quiescent and dividing cells.

Importantly, the adenovirus vectors do not normally integrate into thegenome of the host cell, but they have shown efficacy for transient genedelivery into adult stem cells. These vectors have a series ofadvantages and disadvantages. An important advantage is that they can beamplified at high titers and can infect a wide range of cells(Benihoudet al., 1999; Kanerva and Hemminki, 2005). The vectors are generallyeasy to handle due to their stability in various storing conditions.Adenovirus type 5 (Ad5) has been successfully used in delivering genesin human and mouse stem cells(Smith-Arica et al., 2003). The lack ofadenovirus integration into host cell genetic material can in manyinstances be seen as a disadvantage, as its use allows only transientexpression of the therapeutic gene.

For example in a study evaluating the capacity of mesenchymal stem cellsto undergo chondrogenesis when TGF-beta1 and bone morphogencic protein-2(BMP-2) were delivered by adenoviral-mediated expression, thechondrogenesis was found to closely correlated with the level andduration of the transiently expressed proteins. Transgene expression inall aggregates was highly transient, showing a marked decrease after 7days. Chondrogenesis was inhibited in aggregates modified toexpress >100 ng/ml TGF-beta1 or BMP-2; however, this was partly due tothe inhibitory effect of exposure to high adenoviral loads (Mol Ther.2005 August; 12(2):219-28. Gene-induced chondrogenesis of primarymesenchymal stem cells in vitro. Palmer G D, Steinert A, Pascher A,Gouze E, Gouze J N, Betz 0, Johnstone B, Evans C H, Ghivizzani S C.) Ina second model using rat adipose derived stem cells transduced withadenovirus carrying the recombinant human bone morphogenic protein-7(BMP-7) gene showed promising results for an autologous source of stemcells for BMP gene therapy. However, activity assessed by measuringalkaline phosphatase in vitro was transient and peaked on day 8. Thusthe results were similar to those found in the chondrogenesis model(Cytotherapy. 2005; 7(3):273-81).

Thus for therapies or experiments that do not require stable geneexpression adenovirus vectors may be a good option. An additionalimportant problem in using adenovirus vectors is that they can elicit astrong immune response directed against the engineered cells upontransfer into the host. Clearly this may be important issue whenconsidering the application of engineered cells in a therapeutic setting(J. N. Glasgow et al., Transductional and transcriptional targeting ofadenovirus for clinical applications. Curr Gene Ther. 2004 March; 4(1):1-14). In vitro and in vivo induction of bone formation based on ex vivogene therapy using rat adipose-derived adult stem cells expressingBMP-7, Yang M, Ma Q J, Dang G T, Ma K, Chen P, Zhou C Y.)

Adenovirus vectors based on Ad type 5 have been shown to efficiently andtransiently introduce an exogenous gene via the primary receptor,coxsackievirus, and adenovirus receptor (CAR). However, some kinds ofstem cells, such as MSC and hematopoietic stem cells, apparently cannotbe efficiently transduced with conventional adenovirus vectors based onAd serotype 5 (Ad5), because of the lack of CAR expression. To overcomethis problem, fiber-modified adenovirus vectors and an adenovirus vectorbased on another serotype of adenovirus have been developed. (Mol Pharm.2006 March-April; 3(2):95-103. Adenovirus vector-mediated gene transferinto stem cells. Kawabata K, Sakurai F, Koizumi N, Hayakawa T, MizuguchiH. Laboratory of Gene Transfer and Regulation, National Institute ofBiomedical Innovation, Osaka 5670085, Japan.)

-   -   (ii) Adeno-associated virus

Adeno-Associated viruses (AAV) are ubiquitous, noncytopathic,replication-incompetent members of ssDNA animal virus of parvoviridaefamily (G. Gao et al., New recombinant serotypes of AAV vectors. CurrGene Ther. 2005 June; 5(3):285-97). AAV is a small icosahedral viruswith a 4.7 kb genome. These viruses have a characteristic terminiconsisting of palindromic repeats that fold into a hairpin. Theyreplicate with the help of helper virus, which are usually one of themany serotypes of adenovirus. In the absence of helper virus theyintegrate into the human genome at a specific locus (AAVS 1) onchromosome 19 and persist in latent form until helper virus infectionoccurs(Atchison et al., 1965, 1966). AAV can transduce cell types fromdifferent species including mouse, rat and monkey. Among the serotypes,AAV2 is the most studied and widely applied as a gene delivery vector.Its genome encodes two large opening reading frames (ORFs) rep and cap.The rep gene encodes four proteins Rep 78, Rep 68, Rep 52 and Rep 40which play important roles in various stages of the viral life cycle(e.g. DNA replication, transcriptional control, site specificintegration, accumulation of single stranded genome used for viralpackaging). The cap gene encodes three viral capsid proteins VP1, VP2,VP3(Becerra et al., 1988; Buning et al., 2003). The genomic 3′ endserves as the primer for the second strand synthesis and has terminalresolution sites (TRS) which serve as the integration sequence for thevirus as the sequence is identical to the sequence on chromosome19(Young and Samulski, 2001; Young et al., 2000).

These viruses are similar to adenoviruses in that they are able toinfect a wide range of dividing and non-dividing cells. Unlikeadenovirus, they have the ability to integrate into the host genome at aspecific site in the human genome. Unfortunately, due to their ratherbulky genome, the AAV vectors have a limited capacity for the transferof foreign gene inserts(Wu and Ataai, 2000).

RNA virus vectors

-   -   (i) Retroviruses

Retroviral genomes consist of two identical copies of single strandedpositive sense RNAs, 7-10 kb in length coding for three genes; gag, poland env, flanked by long terminal repeats (LTR) (Yu and Schaffer, 2005).The gag gene encodes the core protein capsid containing matrix andnucleocapsid elements that are cleavage products of the gag precursorprotein. The pol gene codes for the viral protease, reversetranscriptase and integrase enzymes derived from gag pol precursor gene.The env gene encodes the envelop glycoprotein which mediates viralentry. An important feature of the retroviral genome is the presence ofLTRs at each end of the genome. These sequences facilitate theinitiation of viral DNA synthesis, moderate integration of the proviralDNA into the host genome, and act as promoters in regulation of viralgene transcription. Retroviruses are subdivided into three generalgroups: the oncoretroviruses (Maloney Murine Leukemia Virus, MoMLV), thelentiviruses (HIV), and the spumaviruses (foamy virus) (Trobridge etal., 2002).

Retroviral based vectors are the most commonly used integrating vectorsfor gene therapy. These vectors generally have a cloning capacity ofapproximately 8 kb and are generated by a complete deletion of the viralsequence with the exception of the LTRs and the cis acting packagingsignals.

The retroviral vectors integrate at random sites in the genome. Theproblems associated with this include potential insertional mutagenesis,and potential oncogenic activity driven from the LTR. The U3 region ofthe LTR harbors promoter and enhancer elements, hence this region whendeleted from the vector leads to a self-inactivating vector where LTRdriven transcription is prevented. An internal promoter can then be usedto drive expression of the transgene.

The initial studies of stem cell gene transfer in mice raised the hopethat gene transfer into humans would be equally as efficient(O'Connorand Crystal, 2006). Unfortunately gene transfer using availableretroviral vector systems to transfect multi-lineage long-termrepopulating stem cells is still significantly more efficient in themouse. The reduced efficacy of gene transfer in humans, as well as theuncontrolled integration of the retroviral vector represents importanthurdles for the application of these vectors as a treatment modality inthe context of stem cell engineering.

(ii) Lentivirus

Lentiviruses are members of Retroviridae family of viruses (M. Schen etal., Gene transfer into hematopoietic stem cells using lentiviralvectors. Curr Gene Ther. 2002 February; 2(1):45-55.) They have a morecomplex genome and replication cycle as compared to theoncoretroviruses(Beyer et al., 2002). They differ from simplerretroviruses in that they possess additional regulatory genes andelements, such as the tat gene, which mediates the transactivation ofviral transcription (Sodroski et al., 1996) and rev, which mediatesnuclear export of unspliced viral RNA(Cochrane et al., 1990; Emerman andTemin, 1986).

Lentivirus vectors are derived from the human immunodeficiency virus(HIV-1) by removing the genes necessary for viral replication renderingthe virus inert. Although they are devoid of replication genes, thevector can still efficiently integrate into the host genome allowingstable expression of the transgene. These vectors have the additionaladvantage of a low cytotoxicity and an ability to infect diverse celltypes. Lentiviral vectors have also been developed from Simian, Equineand Feline origin but the vectors derived from Human ImmunodeficiencyVirus (HIV) are the most common(Young et al., 2006).

Lentivirus vectors are generated by deletion of the entire viralsequence with the exception of the LTRs and cis acting packagingsignals. The resultant vectors have a cloning capacity of about 8 kb.One distinguishing feature of these vectors from retroviral vectors istheir ability to transduce dividing and non-dividing cells as well asterminally differentiated cells(Kosaka et al., 2004). The lentiviraldelivery system is capable of high infection rates in human mesenchymaland embryonic stem cells. In a study by Clements et al., the lentiviralbackbone was modified to express mono- and bi-cistronic transgenes andwas also used to deliver short hairpin ribonucleic acid for specificsilencing of gene expression in human stem cells. (Tissue Eng. 2006July; 12(7): 1741-51. Lentiviral manipulation of gene expression inhuman adult and embryonic stem cells. Clements M O, Godfrey A, CrossleyJ, Wilson S J, Takeuchi Y, Boshoff C.)

Insert Vector genome Inflammatory Vector capacity(kb) Tropism formExpression potential Efficiency Enveloped Retrovirus 8 Dividing cellsIntegrated Stable Low High only Lentivirus 8 Dividing and IntegratedStable Low High non-dividing Non-enveloped Adeno-associated <5 Dividingand Episomal and Stable Low High virus non-dividing integratedAdenovirus 4-25 Dividing and Episomal Transient High High non-dividing

Non-Viral Gene Delivery Systems

(i) Methods for the Facilitated Integration of Genes

In addition to the viral based vectors discussed above, other vectorsystems that lack viral sequence are currently under development. Thealternative strategies include conventional plasmid transfer and theapplication of targeted gene integration through the use of integrase ortransposase technologies. These represent important new approaches forvector integration and have the advantage of being both efficient, andoften site specific in their integration. Currently three recombinasesystems are available for genetic engineering: cre recombinase fromphage P1(Lakso et al., 1992; Orban et al., 1992), FLP (flippase) fromyeast 2 micron plasmid(Dymecki, 1996; Rodriguez et al., 2000), and anintegrase isolated from streptomyses phage ↑:C31(Ginsburg and Calos,2005). Each of these recombinases recognize specific target integrationsites. Cre and FLP recombinase catalyze integration at a 34 bypalindromic sequence called lox P (locus for crossover) and FRT (FLPrecombinase target) respectively. Phage integrase catalyzessite-specific, unidirectional recombination between two short attrecognition sites in mammalian genomes. Recombination results inintegration when the att sites are present on two different DNAmolecules and deletion or inversion when the att sites are on the samemolecule. It has been found to function in tissue culture cells (invitro) as well as in mice (in vivo).

The Sleeping Beauty (SB) transposon is comprised of two invertedterminal repeats of 340 base pairs each(Izsvak et al., 2000). Thissystem directs the precise transfer of specific constructs from a donorplasmid into a mammalian chromosome. The excision and integration of thetranspo son from a plasmid vector into a chromosomal site is mediated bythe SB transposase, which can be delivered to cells as either in a cisor trans manner(Kaminski et al., 2002). A gene in a chromosomallyintegrated transposon can be expressed over the lifetime of a cell. SBtransposons integrate randomly at TA-dinucleotide base pairs althoughthe flanking sequences can influence integration. While the results todate do not suggest that random insertions of SB transposons representthe same level of risks seen with viral vectors, more data are requiredbefore the system can be safely applied to human trials.

Physical Methods to Introduce Vectors into Cell

-   -   (i) Electroporation

Electroporation relies on the use of brief, high voltage electric pulseswhich create transient pores in the membrane by overcoming itscapacitance. One advantage of this method is that it can be utilized forboth stable and transient gene expression in most cell types. Thetechnology relies on the relatively weak nature of the hydrophobic andhydrophilic interactions in the phospholipid membrane and its ability torecover its original state after the disturbance. Once the membrane ispermeabilized, polar molecules can be delivered into the cell with highefficiency. Large charged molecules like DNA and RNA move into the cellthrough a process driven by their electrophoretic gradient. Theamplitude of the pulse governs the total area that would bepermeabilized on the cell surface and the duration of the pulsedetermines the extent of permeabilization(Gabriel and Teissie, 1997).The permeabilized state of the cell depends on the strength of thepulses. Strong pulses can lead to irreversible permeabilization,irreparable damage to the cell and ultimately cell death. For thisreason electroporation is probably the harshest of gene delivery methodsand it generally requires greater quantities of DNA and cells. Theeffectiveness of this method depends on many crucial factors like thesize of the cell, replication and temperature during the application ofpulse(Rols and Teissie, 1990).

The most advantageous feature of this technique is that DNA can betransferred directly into the nucleus increasing its likelihood of beingintegrated into the host genome. Even cells difficult to transfect canbe stably transfected using this method(Aluigi et al., 2005; Zernecke etal., 2003). Modification of the transfection procedure used duringelectroporation has led to the development of an efficient gene transfermethod called nucleofection. The Nucleofector™ technology, is anon-viral electroporation-based gene transfer technique that has beenproven to be an efficient tool for transfecting hard-to-transfect celllines and primary cells including MSC (Michela Aluigi, Stem Cells Vol.24, No. 2, February 2006, pp. 454-461). Biomolecule-based methods

-   -   (i) Protein transduction domains (PTD)

PTD are short peptides that are transported into the cell without theuse of the endocytotic pathway or protein channels. The mechanisminvolved in their entry is not well understood, but it can occur even atlow temperature(Derossi et al. 1996). The two most commonly usednaturally occurring PTDs are the trans-activating activator oftranscription domain (TAT) of human immunodeficiency virus and thehomeodomain of Antennapedia transcription factor. In addition to thesenaturally occurring PTDs, there are a number of artificial peptides thathave the ability to spontaneously cross the cell membrane(Joliot andProchiantz, 2004). These peptides can be covalently linked to thepseudo-peptide backbone of PNA (peptide nucleic acids) to help deliverthem into the cell.

(ii) Liposomes

Liposomes are synthetic vesicles that resemble the cell membrane. Whenlipid molecules are agitated with water they spontaneously formspherical double membrane compartments surrounding an aqueous centerforming liposomes. They can fuse with cells and allow the transfer of“packaged” material into the cell. Liposomes have been successfully usedto deliver genes, drugs, reporter proteins and other biomolecules intocells(Felnerova et al., 2004). The advantage of liposomes is that theyare made of natural biomolecules (lipids) and are nonimmunogenic.

Diverse hydrophilic molecules can be incorporated into them duringformation. For example, when lipids with positively charged head groupare mixed with recombinant DNA they can form lipoplexes in which thenegatively charged DNA is complexed with the positive head groups oflipid molecules. These complexes can then enter the cell through theendocytotic pathway and deliver the DNA into lysosomal compartments. TheDNA molecules can escape this compartment with the help ofdioleoylethanolamine (DOPE) and are transported into the nucleus wherethey can be transcribed(Tranchant et al., 2004).

Despite their simplicity, liposomes suffer from low efficiency oftransfection because they are rapidly cleared by the reticuloendothelialsystem due to adsorption of plasma proteins. Many methods of stabilizingliposomes have been used including modification of the liposomal surfacewith oligosaccharides, thereby sterically stabilizing the liposomes (Xuet al., 2002).

(iii) Immunoliposomes

Immunoliposomes are liposomes with specific antibodies inserted intotheir membranes. The antibodies bind selectively to specific surfacemolecules on the target cell to facilitate uptake. The surface moleculestargeted by the antibodies are those that are preferably internalized bythe cells so that upon binding, the whole complex is taken up. Thisapproach increases the efficiency of transfection by enhancing theintracellular release of liposomal components. These antibodies can beinserted in the liposomal surface through various lipid anchors orattached at the terminus of polyethylene glycol grafted onto theliposomal surface. In addition to providing specificity to genedelivery, the antibodies can also provide a protective covering to theliposomes that helps to limit their degradation after uptake byendogenous RNAses or proteinases(Bendas, 2001). To further preventdegradation of liposomes and their contents in the lysosomalcompartment, pH sensitive immunoliposomes can be employed(Torchilin,2006). These liposomes enhance the release of liposomal content into thecytosol by fusing with the endosomal membrane within the organelle asthey become destabilized and prone to fusion at acidic pH.

In general non-viral gene delivery systems have not been as widelyapplied as a means of gene delivery into stem cells as viral genedelivery systems. However, promising results were demonstrated in astudy looking at the transfection viability, proliferation anddifferentiation of adult neural stem/progenitor cells into the threeneural lineages neurons. Non-viral, non-liposomal gene delivery systems(ExGen500 and FuGene6) had a transfection efficiency of between 16%(ExGen500) and 11% (FuGene6) of cells. FuGene6-treated cells did notdiffer from untransfected cells in their viability or rate ofproliferation, whereas these characteristics were significantly reducedfollowing ExGen500 transfection. Importantly, neither agent affected thepattern of differentiation following transfection. Both agents could beused to genetically label cells, and track their differentiation intothe three neural lineages, after grafting onto ex vivo organotypichippocampal slice cultures (J Gene Med. 2006 January; 8(1):72-81.Efficient non-viral transfection of adult neural stem/progenitor cells,without affecting viability, proliferation or differentiation. Tinsley RB, Faijerson J, Eriksson P S).

(iv) Polymer-based methods

The protonated e-amino groups of poly L-lysine (PLL) interact with thenegatively charged DNA molecules to form complexes that can be used forgene delivery. These complexes can be rather unstable and showed atendency to aggregate (Kwoh et al., 1999). The conjugation ofpolyethylene glycol (PEG) was found to lead to an increased stability ofthe complexes (Lee et al., 2005, Harada-Shiba et al., 2002). To confer adegree of tissue-specificity, targeting molecules such astissue-specific antibodies have also been employed (Trubetskoy et al.,1992, Suh et al., 2001).

An additional gene carrier that has been used for transfecting cells ispolyethylenimine (PEI) which also forms complexes with DNA. Due to thepresence of amines with different pKa values, it has the ability toescape the endosomal compartment (Boussif et al., 1995). PEG graftedonto PEI complexes was found to reduce the cytotoxicity and aggregationof these complexes. This can also be used in combination with conjugatedantibodies to confer tissue-specificity (Mishra et al., 2004, Shi etal., 2003, Chiu et al., 2004, Merdan et al., 2003).

Implications for Medicine

Stem cells not only have the ability to differentiate into diversetissues, but due to their inherent ability to home to damaged tissue,they have the potential to deliver the expression of therapeutic genesto specific tissue environments. Through the use of molecularengineering approaches, stem cells can be used as vehicles toselectively express genes in areas of defects or need, thereby releasingthe therapeutic product of the transfection only where it is required.Diseases where genetically engineered stem cells might play a role infuture are those where a protein or an entire enzyme is missing ornonfunctional or where certain factors provide improved function in aspecific tissue.

A series of studies using stem cells in therapeutic settings have beenalready been conducted for the treatment of a diverse range of diseasesthat include cancer, neurodegenerative disorders such as Parkinson'sdisease or Alzheimer's disease, ischemic disease of the heart, andmuscle dystrophies.

The transfer of drug resistance genes into hematopoietic stem cellsshows promise for the treatment of a variety of inherited diseases.These include; X-linked severe combined immune deficiency, adenosinedeaminase deficiency, thalassemia.

The combined stem cell and gene therapy approach has the potential forbeing tailored for acquired disorders such as breast cancer, lymphomas,brain tumors, and testicular cancer. In this regard, studies using thecombined approach for the treatment of cancer have been initiated. Thesestudies range from improving the drug resistance of transplantedhematopoietic stem cells to using genetically modified stem cells totarget cancer.

Drug resistance genes have been transferred into hematopoietic stemcells for providing myeloprotection against chemotherapy-inducedmyelosuppression or for selecting hematopoietic stem cells that areconcomitantly transduced with another gene for correction of aninherited disorder. (Cancer Gene Ther., 2005 November; 12(11):849-63.Hematopoietic stem cell gene therapy with drug resistance genes: anupdate. Budak-Alpdogan T, Banerjee D, Bertino J R).

Examples of using stem cells to target cancer include the enhancement ofbystander effect-mediated gene therapy using genetically engineeredneural stem cells for the treatment of gliomas, and using hematopoieticstem cells carrying the gene of ribonuclease inhibitor to target thevasculature of melanomas.

An additional approach for cancer makes use of the ability of stem cellsto be recruited to tumor vasculature and to differentiate intoendothelial-like cells. Depending upon the tumor type, approximately 30%of new vascular endothelial cells in tumors can be derived from bonemarrow progenitors(Hammerling and Ganss, 2006). Thus, the use ofgenetically modified progenitor cells recruited from the peripheralcirculation may represent a potential vehicle for gene therapy oftumors(Reyes et al., 2002). The Herpes simplex virus 1 (HSV) thymidinekinase (tk) suicide gene together with Ganciclovir® (GCV) have beensuccessfully used for the in vivo treatment of various solidtumors(Dancer et al., 2003; Pasanen et al., 2003). The selectiveexpression of HSV-tk by endothelial cells during neovascularization incombination with tk modification of GCV leads to a lethal environmentfor proliferating cells. A series of promoters have been identified thatare induced during neovascularization allowing the selective activationof the suicide gene following the recruitment and differentiation ofengineered precursor cells.

A “bystander effect” is described as the ability of cells to mediatecell damage to distant cells. In a recent study by Li et al. thebystander effect of neural stem cells transduced with the HSV-tk gene(NSCtk) on rat glioma cells was examined. Intracranial co-implantationexperiments in athymic nude mice or Sprague-Dawley rats, showed that theanimals co-implanted with NSCtk and glioma cells and then treated withGanciclovir® (GCV) showed no intracranial tumors and survived more than100 days, while those treated with physiological saline (PS) died oftumor progression. (Cancer Gene Ther., 2005 July; 12(7):600-7. Bystandereffect-mediated gene therapy of gliomas using genetically engineeredneural stem cells. Li S, Tokuyama T, Yamamoto J, Koide M, Yokota N,Namba H).

Human ribonuclease inhibitor (hRI) can inhibit the activity ofpancreatic RNase (RNase A) and it has been suggested that RI may act asa latent antiangiogenic agent. Fu et al. examined the feasibility oftransfecting the RI gene into murine hematopoietic cells and theninducing expression to block angiogenesis in solid tumors. RI from humanplacenta was cloned and inserted into the retroviral vector pLNCX.Murine bone marrow hematopoietic cells were then infected with thepLNCX-RI retroviral vector. Infected cells were then injected intolethally irradiated mice. After administration of hematopoietic cellscarrying the RI gene, the mice were implanted with B 16 melanomas andthe tumor was grown for 21 days. Tumors from the control groups becamelarge and well vascularized. In contrast, tumors from mice treated withhematopoietic cells carrying the RI gene were small and possessed arelatively low density of blood vessels. (Cancer Gene Ther. 2005 March;12(3):268-75. Anti-tumor effect of hematopoietic cells carrying the geneof ribonuclease inhibitor. Fu P, Chen J, Tian Y, Watkins T, Cui X, ZhaoB.)

Many studies that focus on Parkinson's disease use either celltransplantation or gene therapy (Gene Ther., 2003 September; 10(20):1721-7. Gene therapy progress and prospects: Parkinson's disease. BurtonE A, Glorioso J C, Fink D J). However, few studies to date have combinedthe two approaches. Liu et al. used bone marrow derived stromal cells todeliver therapeutic genes to the brain. The authors used anadeno-associated virus (AAV) vector to deliver tyrosine hydroxylase (TH)gene to bone marrow stromal cells. MSCs expressing TH gene were thentransplanted into the striatum of Parkinson's disease rat. The geneexpression efficiency was found to be approximately 75%. Functionalimprovement in the diseased rats was detected after TH-engineered marrowstromal cells engraftment. Histological examination showed that the THgene was expressed around the transplantation points, and that thedopamine levels in the lesioned striatum of the rats were higher than incontrols. Functional improvement of the animals was observed (Brain ResBrain Res Protoc., 2005 May; 15(1):46-51. Epub 2005, Apr. 22.Therapeutic benefit of TH-engineered mesenchymal stem cells forParkinson's disease. Lu L, Zhao C, Liu Y, Sun X, Duan C, Ji M, Zhao H,Xu Q, Yang H.)

Ischemic cardiovascular disease is an additional target for engineeredstem cell therapy. Chen et al., used purified CD34(+) cells obtainedfrom human umbilical cord blood, transfected with human angiopoietin-1(Angl) and VEGF(165) genes using an AAV vector. The engineered cellswere injected together with VEGF intramyocardially at the left anteriorfree wall, which led to decreased infarct size, and significantlyincreased capillary density after treatment, as well as improved longterm cardiac performance measured using echocardiography 4 weeks aftermyocardial infarction. (Eur J Clin Invest., 2005 November;35(11):677-86. Combined cord blood stem cells and gene therapy enhancesangiogenesis and improves cardiac performance in mouse after acutemyocardial infarction. Chen H K, Hung H F, Shyu K G, Wang B W, Sheu J R,Liang Y J, Chang C C, Kuan P.)

The muscle dystrophies represent a heterogeneous group of neuromusculardisorders characterized by progressive muscle wasting. To date noadequate treatment modality exists for these patients. Adult stem cellpopulations, including MSC, as well as embryonic stem cells have beenevaluated for their ability to correct the dystrophic phenotype. Todate, the described methods have not shown much promise. The reasonsdescribed for failure exemplifies the difficulties researchers encounterwhen using genetically modified stem cells: the underlying mechanismresponsible for a myogenic potential in stem cells has not yet beenfully elucidated, homing of the donor population to the muscle is ofteninadequate, and poorly understood immune responses in the recipient canlead to limited treatment success (Stem cell based therapies to treatmuscular dystrophies. Price, Kuroda, Rudnicki) One approach used for thetreatment of Duchenne muscular dystrophy (DMD) utilizes autologous celltransplantation of myogenic stem cells that have been transduced with atherapeutic expression cassette. Development of this method has beenhampered by a series of problems including; a low frequency of cellularengraftment, difficulty in tracing transplanted cells, rapid loss ofautologous cells carrying marker genes, and difficulty in introducingthe stable transfer of the large dystrophin gene into myogenic stemcells.

A mini Dys-GFP fusion gene was engineered by replacing the dystrophinC-terminal domain (DeltaCT) with an eGFP coding sequence and removingmuch of the dystrophin central rod domain (DeltaH2-R19). In a transgenicmdx(4Cv) mouse expressing the miniDys-GFP fusion protein under thecontrol of a skeletal muscle-specific promoter, the green fusion proteinlocalized on the sarcolemma, where it assembled thedystrophin-glycoprotein complex and prevented the development ofdystrophy in transgenic mdx(4Cv) muscles. (Hum Mol Genet., 2006 May 15;15(10):1610-22. Epub 2006 Apr. 4. A highly functionalmini-dystrophin/GFP fusion gene for cell and gene therapy studies ofDuchenne muscular dystrophy. Li S, Kimura E, Ng R, Fall B M, Meuse L,Reyes M, Faulkner J A, Chamberlain J S.)

Wiskott-Aldrich-Syndrome is characterized by thrombocytopenia,dysregulation and propensity towards lymphoma development later in lifeand represents a potential target for engineered stem cell therapy(Dupreet al., 2006). Fanconi anemia, is considered a “stem cell disease” andhas been the subject of intensive research for treatment using genetherapy. This disease represents the best-characterized congenitaldefect of hematopoietic stem cells. It is a rare hereditary diseasecharacterized by bone marrow failure and developmental anomalies; a highincidence of myelodysplasia, acute nonlymphocytic leukemia, and solidtumors. The genetic basis for Fanconi anemia lies in selective mutationsin any one of the known Fanconi anemia genes, making this disease acandidate for gene therapy. But the disease is complex as at least 12genetic subtypes have been described (FA-A, -B, -C, -D1, -D2, -E, -F,-G, -I, -J, -L, -M) and all, with the exception of FA-I have been linkedto a distinct gene. Most FA proteins form a complex that activates theFANCD2 protein via monoubiquitination, while FANCJ and FANCD1/BRCA2function downstream of this step. The FA proteins typically lackfunctional domains, except for FANCJ/BRIP1 and FANCM, which are DNAhelicases, and FANCL, which is probably an E3 ubiquitin conjugatingenzyme. Based on the hypersensitivity to cross-linking agents, the FAproteins are thought to function in the repair of DNA interstrandcross-links, which block the progression of DNA replication forks. (CellOncol. 2006; 28(1-2):3-29. The Fanconi anemia pathway of genomicmaintenance. Levitus M. Joenje H, de Winter J P.

Additional inherited stem cell defects that are potential candidates forgene therapy include amegakaryocytic thrombocytopenia, dyskeratosiscongenity and Shwachman-Diamond syndrome. Thalassemias and Sickle Celldisease belong to the group of hereditary hemolytic anemias thatrepresent the most common inherited diseases worldwide and thus areimportant candidates for stem cell gene therapy(Persons and Tisdale,2004).

A good example using genetically modified mesenchymal stem cells in aclinical setting is the correction of the genetic mutation in thebridled bone disease osteogenesis imperfecta. Osteogenesis imperfectacauses fragile bones due to mutations in the collagen-1-encoding genes,COLIA1 or COLIA2. Chamberlain et al. obtained mesenchymal stem cells(MSCs) from the bones of osteogenesis imperfecta patients and identifiedpoint mutations in the COLIA1 gene(Chamberlain et al., 2004). MSCs weresuccessfully infected with an adeno-associated virus to target anddeactivate the mutated COLIA1 gene. The corrected MSCs were thentransplanted into immunodeficient mice and damaged cells demonstratedimproved stability and collagen processing.

EXAMPLES Example 1

Multipotent adult stem cells are isolated from the bone marrow and othersources of a patient or donor using adherent growth in-vitro todetermine cell activity and biological function. At this in-vitro stagethe adherent growing cell do not express the “stem cell marker” CD34 andare therefore considered CD34-negative during in-vitro culturing. Atthis stage, CD34-negative, adherent growing stem cells are transient orstably transfected by viral or non-viral technologies and expandedselectively in-vitro before in-vivo application. For the generation oftransgenic CD34-negative progenitor cells two promoters are used forselection and organ/target-specific inducibility of the therapeuticgene. The gene transfer system is chosen based on their transfection andintegration (if desired) combined with adhesion selectivity. As thisexample, the tie2-promotor enhancer is driving the HSV-TK gene, which isexpressed only in the context of endothelial differentiation, whichhappens during tumor neo-angiogenesis. While circulating endogenous aswell as systemically administered stem cells are recruitedphysiologically to the site of tumor growth to participate in the tumorneo-angiogenesis (independently whether it is the primary tumor site ormetastasis), the stem cells differentiate into tumor endothelial cells.During this process of organ-specific differentiation, the stem cellsexpress the HSV-TK gene driven by the angiogenesis-related tie2activation. Now the prodrug Ganciclovir® can be given to the patient andis converted by the HSV-TK into the cytotoxic substance at the site oftumor angiogenesis. This approach has been successfully shown inpre-clinical models for breast cancer, metastatic colo-rectal cancer,pancreas carcinoma and glioblastoma. An application can be envisionedfor any (malignant) neoplasia that relies on tumor neo-angiogenesis.This approach aims at the disruption of tumor angiogenesis.

Example 2

As in EXAMPLE 1, but instead of expressing HSV-TK, expressing clottingsubstances as cytotoxic proteins.

Example 3

Angiogenesis is also a pivotal biological process in tissue remodelingand wound healing. This does not only apply for lesions of the skin ormucosa but also for other tissues, like the lack of insulin-producingbeta-cells in the pancreas, leading to Insulin-dependent Diabetesmellitus (IDDM). The systemic application of transgenic CD34-negativeprogenitor cells can also induce the activation of otherwise quiescentislet progenitor cells in the pancreas, replenishing the endocrinepancreas and correcting the state of hyperglycemia in IDDM patients. Thetie-2 enhancer promoter activates the gene for vasoactive substanceslike VEGF promoting tissue remodeling and wound healing.

Example 4

As in EXAMPLE 3, but in combination with the transplantation ofallogeneic islet cells if endogenous regeneration is not sufficientanymore.

Example 5

As in EXAMPLE 1, but transgenic cells with enhanced homing capabilitiesto the site of tumor growth, tissue remodeling or wound healing applyingchemokine biology. CD34-negative, adherent growing stem cells areengineered using GPI-mucin-chemokines. These agents will allow theselective expression of specific chemokines linked to the mucin-domaintaken either from CX3CL1 or CXC16 fused to a GPI anchor. The expressionof these chemokine-mucin agents will recruit complementary leukocytesexpressing the chemokine receptor. For example, CXCL10-mucin-GPIexpression under the control of the tie2 promoter enhancer in thecontext of tumor therapy will facilitate the recruitment of effector Tcells into the tumor environment. This will act as an adjuvant for tumorimmune therapy. The same approach could also be used in tissueremodeling to facilitate the parallel recruitment of select leukocytepopulations.

Example 6

As in EXAMPLE 1, but genetically engineering CD34-negative stem cellsthat can modulate the inflammatory environment, e.g. in autoimmunedisorders like chronic-inflammatory bowel disease or graft-versus-hostdisease after allogeneic bone marrow/stem cell transplantation. This canalso be facilitated by the site-specific expression of anti-inflammatorysubstances like interleukins (IL-10).

Example 7

As in EXAMPLE 1, but with the site-specific expression of common viralantigens e.g. which induce an internal vaccination boost at the site oftumor growth, e.g. measles or chicken pox.

Example 8

As in EXAMPLE 1, but the therapeutic gene activation is suppressed bygenes of an early developmental stage (e.g. Noggin), which eventuallybecomes down regulated during the differentiation of the transgenicprogenitor cells in mature tissue at the site of the tumor or tissueremodeling/regeneration.

Part II Breast and Pancreatic Tumor Models Synopsis

Tumor angiogenesis represents a promising target for the selectivedelivery of cancer therapeutics. Bone marrow-derived mesenchymal stemcells were developed to selectively target exogenous genes to tumorangiogenesis environments. The results of these experiments show thatexogenously added MSC home to tumors where they undergo differentiation.Genes such as the RFP reporter gene as well as the suicide gene HSV-tkare selectively expressed during differentiation under control of theTie2 promoter/enhancer. The administration of the pro-drug Ganciclovir®in concert with tk expression effectively targets the tumor and resultsin the suppression of tumor growth.

Endogenous Mouse Breast Cancer Model

A previously established murine breast cancer model was used by Dr.Christoph Klein to study the use of engineered MSC in tumorangiongenesis. This model is broadly applicable to human breast cancer.In this model, transgenic mice carrying the activated rat c-neu oncogeneunder transcriptional control of the MMTV promoter have been backcrossedto BALB/c mice with the aim of developing a broadly applicable model forcancer therapy. Female HER-2/neu (neu-N) transgenic mice, which expressthe nontransforming rat proto-oncogene, develop spontaneous focalmammary adeno-carcinomas beginning at 5-6 months of age. The developmentand histology of these tumors bear resemblance to what is seen inpatients with breast cancer.

Expression of RFP and GFP Genes in imMSC Under Control of EndothelialSpecific Promoters in the Context of Tumor Angiogenesis

To assess the control of tissue specific expression gene expression inimMSC in the context of tumor angiogenesis, and to follow thedistribution of imMSCs over longer time periods microscopically, red andgreen fluorescent protein (RFP, GFP) genes have been cloned intomodified expression vectors to detect in vivo.

Mice

Female HER-2/neu (neu-N) transgenic mice expressing the nontransformingrat proto-oncogene, are known to develop spontaneous focal mammaryadeno-carcinomas at 5-6 months of age. BALB-neuT transgenic mice weremaintained in accordance with the Agreement to the European UnionGuidelines. Mice were screened for hemizygosity (neuT+/neuT−). Mammaryglands of Balb-neuT female mice were inspected twice a week and arisingtumors were measured.

The genetically modified cells were injected into the breast cancermodel mice following surgical resection of the primary tumor. As theresidual tumor grew back, the exogenously added imMSC provided precursorcells for neovascularization. The Tie-2-RFP (endothelial specificpromoter driving red florescence protein) stably transfected imMSCs werefound to readily home to the tumor, differentiate to endothelial cells,and express the RFP reporter gene (FIG. 5). In these experiments theintegration of the mesenchymal cells into the primary mammary tumors ofBalb-neuT mice was evaluated. After three applications ofRFP-transfected, RFP-expres sing cells could be detected in vessel-likeregions in all sections. The results demonstrate that the cells home tosites of neovascularization and express marker genes through theactivated Tie2 promoter/enhancer.

Inhibiting Tumor Growth by Targeting a Suicide Gene in the Endothelium

The protocol was then altered to evaluate the effect of the suicide geneHSV-tk. The tk gene product in combination with the prodrug Ganciclovir®(GCV) produces a potent toxin which affects replicative cells. ImMSCswere stably transfected with plasmids carrying the herpes simplexvirus-thymidine kinase (tk) gene driven by the vascular endothelial Tie2promoter/enhancer. To this end, the murine model of breast cancerangiogenesis was used again to evaluate the engineered MSC line inanti-angiogenesis therapy.

Approach I. Injection of Engineered MSC and Ganciclovir® Treatment inthe Phase of Exponential Tumor Growth at the Age of 18 or 22 Weeks

The Treatment of Balb-neuT Trsg

Mice started on day 0 (week 22), with injection of 0.2 ml cells (500,000cells) and 0.2 ml PBS as control. On days 5 to 8, Ganciclovir® wasapplied in a daily dose of 30 μg/g BW, e.g. 100 μl for a mouse with 21 gBW. After day 9, mice treatment cycles were repeated until dissection.During the treatment tumor progression, bodyweight (measured on day 0and 6 of each therapy cycle) and behavior were recorded.

To get an overall impression of the effect of the treatment withTie2-Tk-as-transfected cells and GCV respectively compared with bothcontrol groups, the macroscopic value of bodyweight was recorded duringtreatment until dissection. Measure points were days 0 and 5 of eachcycle of therapy and the day of dissection. The experiments included onetreatment and two control groups of mice and started at two differenttime points, 18 and 22 weeks of age.

TABLE 2 Data of treatment groups after dissection, including bodyweight,absolute and relative tumor load. Absolute Relative Treatment AmountBodyweight tumorload tumorload group of mice [g] SD [g] SD [g] SDPBS-22- 2 32.6 0.8 8.5 1 0.265 0.035 RFP-22- 3 29.4 3 8.3 0.9 0.2870.032 Tk-as/ 3 31.8 1.1 8.1 0.9 0.257 0.021 GCV-22-

Approach II. Evaluation of Therapy in the Context of Tumor RegrowthFollowing Surgical Resection

In patients following the surgical removal of a tumor, residual tumorthat is missed during surgery often grows out, leading to a reoccurrenceof cancer. To test the efficacy of the engineered MSC/tk therapy in thiscontext, the breast tissue from Balb/c neu-N transgenic mice wasresected at 18 weeks of age, leading to a delayed onset of primarytumor. Following surgery, the mice were treated with the MSC-tk and GCVregimen as described above. The treatment resulted in a dramaticreduction in tumor growth in the treated mice (FIG. 8).

Pancreatic Tumor Model

An orthotopic pancreatic carcinoma model was then developed in C57BI/6mice to assess the efficacy of the MSC-based therapy in a differenttumor system. The system had been previously established by ChristianeBruns and Claudius Conrad (Surgery Department, LMU). In this model,Panc02 pancreatic carcinoma cells syngeneic to C57BI/6 mice wereinjected subcapsularly in a region of the pancreas just beneath thespleen to create primary pancreatic tumors. Constructs with GFP underthe control of the CMV promoter, and RFP and tk under the control ofTie2 promoter/enhancer, were introduced into the MSC isolated fromC57BI/6 mice. The transfected stem cells were given systemically viai.v. injections. In a preliminary experiment, MSC engineered toconstitutively express GFP (under control of the CMV promoter) wereinjected into mice with growing tumors. The cells were found toefficiently home to the tumors (FIG. 9).

In parallel experiments, mice with growing tumors were injected with theTie2-RFP engineered MSC. After five days, the animals were sacrificedand the tumors were examined for expression of RFP. The results show astrong upregulation of RFP in the context of tumor (FIG. 9). RFP was notdetected in other organs (spleen, lymph nodes and thymus).

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What is claimed is:
 1. A method for treating a subject afflicted with agastrointestinal disorder comprising introducing into the subject'sbloodstream a therapeutically effective number of genetically modifiedCD34− stem cells, wherein (a) each of the genetically modified CD34−stem cells contains an exogenous nucleic acid comprising (i) a regionencoding a protein which enhances endothelial cell growth, which regionis operably linked to (ii) an endothelium-specific promoter orpromoter/enhancer combination, (b) the introduction of the geneticallymodified CD34− stem cells is not preceded, accompanied or followed bymyeloablation, and (c) the gastrointestinal disorder is characterized bya need for cell proliferation in the gastrointestinal endothelium. 2.The method of claim 1, wherein the subject is human.
 3. The method ofclaim 2, wherein the gastrointestinal disorder is colitis, ulcerativecolitis, inflammatory bowel disorder or Crohn's disease.
 4. The methodof claim 2, wherein the promoter/enhancer combination is the Tie2promoter/enhancer and the protein which enhances endothelial cell growthis a vascular endothelial growth factor (VEGF).
 5. The method of claim2, wherein the genetically modified CD34− stem cells are allogenic withrespect to the subject.
 6. The method of claim 2, wherein thegenetically modified CD34− stem cells are autologous with respect to thesubject.
 7. The method of claim 2, wherein the therapeutically effectivenumber of genetically modified CD34− stem cells is from about 1×10³ toabout 1×10⁷ cells/kg body weight.